Bottom Line:
Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines.Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells.Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.

Figure 5: Chicken myotubes display a more vigorous cytokine response to avian influenza virus infection than do duck myotubes. (A and B) Chicken myotubes infected with a LPAI H2N3 virus showed a strong induction of IFN-α (40-fold induction) and a moderate induction of proinflammatory cytokines (LITAF, IL-6, and IL-8) (A); the corresponding duck muscle cells showed weaker induction, with progressive downregulation of the TNF-α response (B). (C and E) With HPAI H5N1 50-92 (C) or HPAI H5N1 tyTy05 (E) virus, the level of IFN-α mRNA induction was not >10-fold, while IL-8 mRNA was more strongly induced in chicken than in duck muscle cells. (D and F) In duck cells, TNF-α gene transcription was downregulated by both HPAI H5N1 viruses, unlike LITAF gene induction in the corresponding chicken cells (C and E). Duck IFN-α mRNA induction was greater with HPAI H5N1 tyTy05 virus (F) than with HPAI H5N1 50-92 virus (D). (G) Duck myotubes infected with LPAI H2N3 and HPAI H5N1 50-92 viruses showed modest upregulation of the viral RNA sensor RIG-I, but with HPAI H5N1 tyTy05 virus, RIG-I expression was downregulated. mRNA levels were normalized to the 18S rRNA gene and are expressed as fold changes in relation to uninfected controls at each p.i. time point. The fold change for each gene is the mean of data from three biological replicates, with error bars indicating standard deviations. A significant increase or decrease in mRNA levels between 6 h p.i. and later times of infection was determined by a two-sample unpaired t test (*, P < 0.05; **, P < 0.01).

Mentions:
Following the findings of extensive cell death associated with prominent apoptotic changes in infected chicken and duck myotubes, we examined the expression of IFN-α and the proinflammatory cytokines TNF-α, IL-6, and IL-8 in these infected cells (MOI of 1.0 over 24 h). The IFN-α gene transcript was strongly upregulated (≈40-fold increase) in differentiated chicken muscle cells at 24 h p.i. with LPAI H2N3 virus (Fig. 5A). Additionally, levels of the proinflammatory cytokines LITAF (TNF-α), IL-6, and IL-8 were clearly upregulated at 12 h and 24 h p.i. in chicken muscle cells. In contrast, the corresponding duck muscle cells displayed only a modest induction of IFN-α, IL-6, and IL-8 and a progressive downregulation of TNF-α (Fig. 5B). Next, chicken myotubes were infected with HPAI H5N1 50-92 virus at an MOI of 1.0. Unexpectedly, this HPAI virus elicited a less vigorous cytokine response than did the LPAI H2N3 virus; in particular, the level of IFN-α induction was <10-fold at 24 h p.i. (Fig. 5C). In duck myotubes, HPAI H5N1 50-92 virus infection also induced a weak cytokine response, with a notable downregulation of TNF-α at 24 h p.i. (Fig. 5D). Chicken myotubes infected with HPAI H5N1 tyTy05 virus did not exceed 10-fold IFN-α induction at 24 h p.i. (Fig. 5E). Among the four proinflammatory cytokines, IL-8 was most strongly induced (40-fold increase). Duck myotubes infected with the same high-pathogenicity virus displayed a weak proinflammatory response, with downregulation of TNF-α and IL-6 at 24 h of infection. Duck IFN-α showed a 25-fold induction (Fig. 5F). On the whole, chicken myotubes appeared to be more proinflammatory in response to LPAI and HPAI viruses than did the corresponding duck myotubes.

Figure 5: Chicken myotubes display a more vigorous cytokine response to avian influenza virus infection than do duck myotubes. (A and B) Chicken myotubes infected with a LPAI H2N3 virus showed a strong induction of IFN-α (40-fold induction) and a moderate induction of proinflammatory cytokines (LITAF, IL-6, and IL-8) (A); the corresponding duck muscle cells showed weaker induction, with progressive downregulation of the TNF-α response (B). (C and E) With HPAI H5N1 50-92 (C) or HPAI H5N1 tyTy05 (E) virus, the level of IFN-α mRNA induction was not >10-fold, while IL-8 mRNA was more strongly induced in chicken than in duck muscle cells. (D and F) In duck cells, TNF-α gene transcription was downregulated by both HPAI H5N1 viruses, unlike LITAF gene induction in the corresponding chicken cells (C and E). Duck IFN-α mRNA induction was greater with HPAI H5N1 tyTy05 virus (F) than with HPAI H5N1 50-92 virus (D). (G) Duck myotubes infected with LPAI H2N3 and HPAI H5N1 50-92 viruses showed modest upregulation of the viral RNA sensor RIG-I, but with HPAI H5N1 tyTy05 virus, RIG-I expression was downregulated. mRNA levels were normalized to the 18S rRNA gene and are expressed as fold changes in relation to uninfected controls at each p.i. time point. The fold change for each gene is the mean of data from three biological replicates, with error bars indicating standard deviations. A significant increase or decrease in mRNA levels between 6 h p.i. and later times of infection was determined by a two-sample unpaired t test (*, P < 0.05; **, P < 0.01).

Mentions:
Following the findings of extensive cell death associated with prominent apoptotic changes in infected chicken and duck myotubes, we examined the expression of IFN-α and the proinflammatory cytokines TNF-α, IL-6, and IL-8 in these infected cells (MOI of 1.0 over 24 h). The IFN-α gene transcript was strongly upregulated (≈40-fold increase) in differentiated chicken muscle cells at 24 h p.i. with LPAI H2N3 virus (Fig. 5A). Additionally, levels of the proinflammatory cytokines LITAF (TNF-α), IL-6, and IL-8 were clearly upregulated at 12 h and 24 h p.i. in chicken muscle cells. In contrast, the corresponding duck muscle cells displayed only a modest induction of IFN-α, IL-6, and IL-8 and a progressive downregulation of TNF-α (Fig. 5B). Next, chicken myotubes were infected with HPAI H5N1 50-92 virus at an MOI of 1.0. Unexpectedly, this HPAI virus elicited a less vigorous cytokine response than did the LPAI H2N3 virus; in particular, the level of IFN-α induction was <10-fold at 24 h p.i. (Fig. 5C). In duck myotubes, HPAI H5N1 50-92 virus infection also induced a weak cytokine response, with a notable downregulation of TNF-α at 24 h p.i. (Fig. 5D). Chicken myotubes infected with HPAI H5N1 tyTy05 virus did not exceed 10-fold IFN-α induction at 24 h p.i. (Fig. 5E). Among the four proinflammatory cytokines, IL-8 was most strongly induced (40-fold increase). Duck myotubes infected with the same high-pathogenicity virus displayed a weak proinflammatory response, with downregulation of TNF-α and IL-6 at 24 h of infection. Duck IFN-α showed a 25-fold induction (Fig. 5F). On the whole, chicken myotubes appeared to be more proinflammatory in response to LPAI and HPAI viruses than did the corresponding duck myotubes.

Bottom Line:
Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines.Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells.Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.